The present invention relates generally to the field of diagnostic tests and, more particularly, to a unitary reagent test for determining uric acid in a fluid sample.
In primate metabolism, there is a constant endogenous conversion of ingested nucleoproteins to purines and pyrimidines. The purines, by a catabolic process, then undergo further deamination and partial oxidation to uric acid, which in man is normally excreted in the urine. Thus, a nominal concentration of uric acid is present in human blood and urine at all times.
Ingestion of purine-containing food normally has no affect on the uric acid blood serum content, except in the case of renal insufficiency, in which event the concentration is elevated. In certain other pathological conditions not associated with dietary ingestion of purine-containing food, for example uremia and gout, there is an abnormal increase in the amount of uric acid found in the blood serum. Also, uric acid concentration in the serum is elevated in conditions associated with excessive destruction of leucocyte nuclei, such as leukemia and pneumonia.
A serum uric acid test has been recognized as useful as an aid in diagnosing the foregoing conditions and, in some instances, distinguishing between closely related abnormal conditions, for example, gout and arthritis. Gout is characterized by an abnormal increase in blood serum uric acid, whereas arthritis does not exhibit such increase. It is therefore desirable to provide a simple and economical test which affords a precise and specific determination of the concentration of uric acid in blood serum.
Uric acid is normally found in blood serum in quantities from about 0.7 to about 6.0 milligrams per 100 ml of blood serum, generally reported as milligrams percent (mg%). In the abnormal conditions enumerated above, the uric acid content in the blood serum often attains values of 10 mg% or higher.
The prior art has disclosed a number of methods for determining uric acid in blood serum. Among the more widely used conventional methods are colorimetric procedures utilizing blood filtrates. Some of these procedures depend upon the precipitation of uric acid from the blood filtrate, for example, as a silver salt, and the formation therewith of a chromogenic adduct by reaction with either a phosphotungstate or arsenotungstate. Other methods utilizing the blood filtrate depend upon the direct treatment of the filtrate with a tungstic acid in the presence of a cyanide-urea solution to develop a color which is then measured using conventional techniques for quantitative estimation of uric acid concentration.
More recently, methods have been proposed which involve the catalyzed oxidation of uric acid to allantoin and hydrogen peroxide. This oxidation is usually accomplished in the presence of atmospheric oxygen and utilizes a material having uricase activity, the reaction occurring at or near pH 9. In such methods a spectrophotometer can be employed to measure the disappearance of the characteristic uric acid spectrum during its conversion to allantoin and hydrogen peroxide. Another method utilizes a colorimetric means for measuring the hydrogen peroxide produced in stoichiometric amounts during such degradation of uric acid. See, for example, Albaum U.S. Pat. No. 3,349,006 and Wachter U.S. Pat. No. 3,335,069 (both assigned to the assignee of the present invention).
In the enzymatic conversion test, where the amount of hydrogen peroxide produced is directly proportional to the amount of uric acid present in the blood serum, the hydrogen peroxide is detected by means of a color change produced upon oxidation of a color forming substance in the presence of a substance having peroxidative activity. This reaction occurs at acid pH. A catalase inhibitor, such as sodium azide or sodium cyanide, has usually been required to prevent catalase destruction of the peroxide. The color obtained is then compared visually to standards, or measured electronically, to give a quantitative estimation of uric acid present in the fluid being tested.
French Pat. No. 72/31557 discloses a fundamentally different enzyme catalyzed reaction wherein catalase and aldehyde-free methanol are added along with the uricase to the sample (buffered to pH 8), and 3-methyl-2-benzothiazolinone hydrazone (buffered to pH 3) and FeCl.sub.3 in HCl are then added to give a blue coloration.
Kano U.S. Pat. No. 3,862,885 discloses a process for uric acid determination by generating hydrogen peroxide with a microbe-originated uricase and a catalase-inhibitor (buffered to pH 5.5-7.0) and measuring the peroxide generated in the presence of an anionic surface active agent, a chromogen and peroxidase (pH 4.0-7.0).
Gochman and Schmitz have reported using 3-methyl-2-benzothiazolinone hydrazone hydrochloride with N,N-dimethylaniline to form an azo dye indicator in automated determinations of uric acid, Clin. Chem 17: 1154 (1971).
Notwithstanding the contributions by prior workers in the field, these procedures have had the disadvantage of requiring a series of separate operations, usually carried out in liquid phase. Coupled reactions using animal uricase and peroxidase simultaneously have been considered impossible because of the competition between uric acid and the chromogen, both being oxidized by H.sub.2 O.sub.2 in the presence of peroxidase, and of the drastic difference in the optimum pH of the two enzymes used. The prior art systems where both reactions are carried out at or near the same pH have the further drawback that certain reaction component candidates, such as animal-originated uricase, which exhibit superior performance characteristics cannot be used therein because they are inoperable as a component of the peroxidase-generating system within the pH range required by the reaction.
Thus, incorporation of means for uric acid determination in a stabilized, unitary reagent test has heretofore been impossible.